Any mutagenesis assay aimed at elucidating the factors leading to an increased incidence of cancer and purporting to play a relevant role in cancer risk assessment should preferably be applicable to several species, i.e., mouse, rat and human. The importance of apoptosis, or "programmed cell death", for multicellular organisms is well established. One key apoptotic pathway in vertebrates is triggered when the mitochondrial protein cytochrome c is released from mitochondria in response to DNA damage. The cytochrome c gene has been evolutionarily conserved from nematodes to man and could serve as the basis of a eukaryotic mutagenesis assay used for human cancer risk assessment, drug development and toxicity testing, and the extrapolation of mutagenicity data between species. The aims of the current application are to optimize the experimental protocols for the new cytochrome c mutation assay in order to achieve a robust, reproducible, easy-to-use and high throughput platform and to validate the new assay using untreated and treated tissues and cell lines of murine and human origin. It is anticipated that the validation of this new in vivo model will provide a unique opportunity to study in vivo mutant frequencies in human tissues and cell lines established from human tissues.